Advances in the treatment of lung cancer will require better understanding of its molecular pathogenesis. Aberrant differentiation is one of the hallmarks of cancer, and the contribution of differentiation arrest to the multi-step carcinogenesis process has been widely accepted but poorly characterized. The transcriptional control of differentiation pathways in airway epithelium is poorly understood, and its abnormalities in lung cancer are largely unknown. The long range goals of this project are to further our understanding of disruption of normal differentiation pathways in lung cancer, to gain further insight into the molecular steps leading to lung cancer by identifying critical transcription factor pathways involved in this process, and to evaluate the expression levels of these differentiation factors as potential prognostic tools and biomarkers using primary lung cancer specimens. Previous studies demonstrated that the transcription factor CCAAT Enhancer Binding Protein Alpha (C/EBP alpha) is critical for normal granulocyte differentiation, and abnormalities of C/EBP alpha, including mutations and decreased expression were found in granulocytic leukemias, defining C/EBP alpha as a true tumor suppressor in hematopoietic neoplasms. Recent studies demonstrated that C/EBP alpha expression is downregulated in approximately half of lung cancer specimens. Furthermore, re-establishment of C/EBP alpha expression in lung cancer cell lines led to proliferation arrest, differentiation, and apoptosis, suggesting that it might play a tumor suppressor role in lung cancer, as well. In order to further test the hypothesis that disruption of C/EBP alpha pathways play a critical role in certain lung cancer types, we propose the following specific aims: (1) To further analyze the role of C/EBP alpha in lung cancer through investigation of other mechanisms of inactivation, including mutational analysis, investigation of loss of heterozygosity (LOH), promoter methylation, and transcriptional repression; (2) To analyze the role of C/EBP alpha target genes in normal lung development and in lung cancer, focusing on the HNF3 beta transcription factor; and (3) To perform clinical correlative studies of C/EBP alpha and its target genes in primary lung cancer specimens. These studies will provide information concerning the molecular pathogenesis of non-small cell lung cancer, the role of differentiation factors in defining prognosis, and identify novel markers of differentiation.